The disclosure concerns the use of nucleotide analogues to provide improved properties to hybridization probes, including DNA and RNA probes and modified nucleic acid probes, such as peptide nucleic acids (PNAs), and to chimeric probes containing two or more types of nucleic acid and/or modified nucleic acid.
Hybridization analysis is central to a variety of techniques in molecular biology and diagnostics, including gene cloning, gene identification, forensic analysis, pharmacogenomics and identification of genetic polymorphisms. Hybridization can be used as an endpoint of an assay, whereby the presence of hybridized probe constitutes the readout for the assay; or hybridization can be used as an initial step in an assay, wherein an event subsequent to hybridization (such as, for example, extension of a hybridized primer or hydrolysis of a hybridized probe) is used as the readout.
Traditionally, hybridization probes and primers have been DNA molecules; however, there are certain disadvantages to the use of DNA as a probe or primer. For example, the base composition of a DNA molecule can affect its effectiveness as a probe or primer in several ways. A DNA molecule with a high concentration of G residues is often difficult to handle (e.g., problems with aggregation and poor solubility) and can yield high background in hybridization reactions. It is also well-known that G-rich DNA molecules are prone to the production of artifacts in the analysis of DNA sequences by gel electrophoresis, presumably due to the adoption of secondary structure by such molecules, despite the denaturing conditions under which such analyses are conducted.
Various modified forms of DNA and DNA analogues have been used in attempts to overcome some of the disadvantages of the use of DNA molecules as probes and primers. Among these are peptide nucleic acids (PNAs, also known as polyamide nucleic acids). Nielsen et al. (1991) Science 254:1497-1500. PNAs contain heterocyclic base units, as found in DNA and RNA, that are linked by a polyamide backbone, instead of the sugar-phosphate backbone characteristic of DNA and RNA. PNAs are capable of hybridization to complementary DNA and RNA target sequences and, in fact, hybridize more strongly than a corresponding nucleic acid probe. Furthermore, PNAs are resistant to many types of nuclease which attack the sugar-phosphate DNA and RNA backbones. Additional advantages of PNAs include the ability of specifically modified PNAs to cross the blood-brain-barrier and the observation that PNAs injected intrathecally can mediate antisense affects in vivo. During et al. (1999) Nature Biotechnol. 17:753-754.
The synthesis of PNA oligomers and reactive monomers used in the synthesis of PNA oligomers have been described in U.S. Pat. Nos. 5,539,082; 5,714,331; 5,773,571; 5,736,336 and 5,766,855. Alternate approaches to PNA synthesis and monomers for PNA synthesis have been summarized. Uhlmann et al. (1998) Angew. Chem. Int. Ed. 37:2796-2823.
However, as they become more widely used, disadvantages of PNAs are also becoming apparent. For example, long PNA oligomers, depending on their sequence, are prone to aggregation, difficult to purify and difficult to characterize. In addition, purine-rich PNA oligomers tend to aggregate and are poorly soluble in aqueous media. Gangamani et al. (1997) Biochem. Biophys. Res. Comm. 240:778-782; Egholm, Cambridge Healthtech Institute""s Seventh Annual Nucleic Acid-Based Technologies, Jun. 21-23, 1999, Washington, D.C.; Uhlmann, Cambridge Healthtech Institute""s Seventh Annual Nucleic Acid-Based Technologies, Jun. 21-23, 1999, Washington, D.C. Consequently, effective use of PNAs in hybridization is limited to sequences in which there are no more than 4-5 consecutive purines, no more than 6 purines in any 10-base portion of the sequence, and/or no more than 3 consecutive G residues. See, for example, http://www.resgen.com/perseptivedesign.html. Furthermore, since PNA-PNA interactions are even stronger than PNA-DNA interactions, PNA-containing probes and primers containing self-complementary sequences cannot generally be used for hybridization to a target sequence. Another consequence of the very strong interaction between PNAs and complementary DNA and/or RNA molecules is that it is difficult to obtain single nucleotide mismatch discrimination using PNA probes. Demidov et al. (1995) Proc. Natl. Acad. Sci. USA 92:2637-2641.
Uhlmann et al., supra reviewed approaches for increasing the solubility of PNAs, including synthesis of PNA/DNA chimeras and addition of terminal lysine residues to a PNA oligomer. They did not disclose the use of nucleotide analogues to increase solubility and improve hybridization properties of PNA oligomers.
Similar design constraints are required in the synthesis of non-PNA-containing oligonucleotide probes and primers. See, for example, the publication entitled xe2x80x9cSequence Detection Systems Quantitative Assay Design and Optimization,xe2x80x9d PE Biosystems, Stock No. 117MI02-01. In these cases, the G/C content of an oligomer must be kept within the range of 20-80% and runs of an identical nucleotide, particularly guanine (G), should be avoided. In particular, the aforementioned publication advises against stretches of four or more G residues and against the presence of a G residue at the 5xe2x80x2 end of a 5xe2x80x2-fluorescently labeled probe. In the case of primers, the five nucleotides at the 3xe2x80x2 end should comprise no more than two G and/or C residues.
The synthesis of pyrazolo[3,4-d]pyrimidine and 7-deazapurine nucleosides, as well as their phosphoramidite monomers for use in oligomer synthesis, have been described. Seela et al (1985) Nucl. Acids Res. 13:911-926; Seela et al. (1988a) Helv. Chim. Acta 71:1191-1198; Seela et al. (1988b) Helv. Chim. Acta 71:1813-1823; and Seela et al. (1987) Biochem. 26:2232-2238. Pyrazolo[3,4-d]pyrimidine and 7-deazapurine nucleosides for use in DNA sequencing and as antiviral agents are disclosed in EP 286 028. Co-owned PCT publication WO 99/51775 discloses the use of pyrazolo[3,4-d]pyrimidine containing oligonucleotides for hybridization and mismatch discrimination. It has been reported that incorporation of 2xe2x80x2-deoxy-7-deazaguanosine into DNA eliminates band compression in GC-rich stretches during DNA sequence analysis by gel electrophoresis (U.S. Pat. No. 5,844,106), decreases tetraplex formation by G-rich sequences (Murchie et al. (1994) EMBO J. 13:993-1001) and reduces formation of aggregates characteristic of DNA molecules containing 2xe2x80x2-deoxyguanosine (U.S. Pat. No. 5,480,980). However, substitution of oligonucleotides with either 7-deazaadenosine (in place of A) or 7-deazaguanosine (in place of G) lowers the Tm of hybrids formed by such substituted oligonucleotides, with greater than one degree reduction in Tm per substituted base. Seela et al. (1987) supra; and Seela et al. (1986) Nucl. Acids Res. 14:2319-2332.
On the other hand, stabilization of duplexes by pyrazolopyrimidine base analogues has been reported. Seela et al. (1988) Helv. Chim. Acta. 71:1191-1198; Seela et al. (1988) Helv. Chim. Acta. 71:1813-1823; and Seela et al. (1989) Nucleic Acids Res. 17:901-910. Oligonucleotides in which one or more purine residues have been substituted by pyrazolo[3,4-d]pyrimidines display enhanced duplex- and triplex-forming ability, as disclosed, for example, in Belousov et al. (1998) Nucleic Acids Res. 26:1324-1328; U.S. Pat. No. 5,594,121 and co-owned PCT publication WO 98/49180. Pyrazolo[3,4-d]pyrimidine residues in oligonucleotides are also useful as sites for attachment of various pendant groups to oligonucleotides. See co-owned PCT Publication WO 90/14353, Nov. 29, 1990 and U.S. Pat. No. 5,824,796. None of these references disclose the use of pyrazolopyrimidines or any other type of base analogue for reducing aggregation and/or increasing solubility of an oligomer, or for decreasing quenching of a fluorophore conjugated to an oligomer.
Conjugates comprising a minor groove binder (MGB), an oligonucleotide wherein one or more purine residues are substituted by a pyrazolo[3,4-d]pyrimidine (PZP) residue, a fluorophore and a fluorescence quencher have been disclosed in co-owned PCT publications WO 99/51621 and WO 99/51775. Such conjugates are used, among other things, as hybridization probes, primers and hydrolyzable probes in 5xe2x80x2-nuclease-based amplification assays. Inclusion of a MGB in these conjugates increases the stability of hybrids formed by the oligonucleotide portion of the conjugate, allowing the design of shorter probes. In addition, both the MGB and the PZP contribute to the ability of such conjugates to exhibit enhanced mismatch discrimination. Neither of the aforementioned publications disclose the use of PZPs or any other type of base analogue for reducing aggregation and/or increasing solubility of an oligomer, or for decreasing quenching of a fluorophore conjugated to an oligomer.
Oligomers wherein at least one of the subunits comprises a pyrazolopyrimidine and/or a pyrrolopyrimidine base analogue are provided. The oligomers can comprise DNA, RNA, PNA, or any combination or chimera thereof. Any number of purine residues in the oligomer can be substituted by a base analogue. Any of the above-mentioned oligomers can comprise additional moieties such as fluorophores, fluorescence quenchers and/or minor groove binders.
Oligomers wherein at least one of the subunits comprises a pyrazolopyrimidine and/or a pyrrolopyrimidine base analogue, when used for hybridization, are less prone to aggregation and self-association, are more soluble, are capable of enhanced mismatch discrimination, and do not quench the emission of conjugated fluorescent labels.
Oligomers comprising one or more PNA residues wherein at least one of the PNA residues comprises a pyrazolopyrimidine and/or a pyrrolopyrimidine base analogue are also provided. The oligomers can comprise exclusively PNA residues, or the oligomers can comprise both PNA and/or DNA and/or RNA nucleotide residues to constitute a PNA/DNA, PNA/RNA or PNA/DNA/RNA chimera. Any number of purine residues in the oligomer can be substituted by a base analogue. Any of the above-mentioned oligomers can comprise additional moieties such as fluorophores, fluorescence quenchers and/or minor groove binders.
In another embodiment, compositions comprising a polymer and a fluorophore are provided, wherein one or more purine-containing residues of the polymer are substituted with a residue comprising a pyrazolopyrimidine and/or pyrrolopyrimidine base analogue. Polymers can comprise PNA, DNA, RNA or any combination or chimera thereof, and the base analogue can be present in any of the PNA, DNA or RNA portions of a chimeric polymer. Any number of purine residues in the polymer can be substituted by a base analogue, in any of the PNA, DNA and/or RNA portions. The above-mentioned compositions can optionally comprise a fluorescence quencher and/or a minor groove binder.
In the polymer-fluorophore compositions just described, quenching of the fluorophore by purine residues in the polymer is reduced when one or more purines are substituted with a base analogue. Such compositions additionally comprising a fluorescence quencher are useful, for example, as probes in hydrolyzable probe assays, in which quenching of the fluorophore by the fluorescence quencher is relieved by hybridization-dependent hydrolysis of probe. The reduction in quenching afforded by substitution of a base analogue for a purine leads to higher fluorescence output after hydrolysis and, hence, greater sensitivity in such assays.
New intermediates for the synthesis of PNA-containing oligomers comprising base analogues are also provided. In one embodiment, acetic acid derivatives of pyrazolopyrimidine and pyrrolopyrimidine base analogues, wherein N1 of the pyrazolopyrimidine or pyrrolopyrimidine is linked to C2 of an acetic acid moiety and functional groups are blocked, are provided. These derivatives are useful for preparation of monomers for automated synthesis of substituted PNAs and PNA/DNA chimeras. Preferred embodiments of these intermediates include 2-{6-[(1E)-1-aza-2-(dimethylamino)vinyl]-4-hydroxypyrazolo[5,4-d]pyrimidinyl}acetic acid; 2-(6-amino-4-hydroxypyrazolo[5,4-d]pyrimidinyl)acetic acid; and 2-(-4-aminopyrazolo[5,4-d]pyrimidinyl)acetic acid.
Also provided are aminoethylglycyl derivatives of the aforementioned acetic acid derivatives of pyrazolopyrimidine and pyrrolopyrimidine base analogues, wherein the xcex1-amino group of a blocked glycyl moiety is derivatized to acetic acid C1 of the acetate and to C2 of an ethylamine moiety. These derivatives are also known as xe2x80x9cPNA monomers.xe2x80x9d Such compounds are useful for automated synthesis of the aforementioned oligomers and polymers. Preferred embodiments of PPG-containing PNA monomers (also known as PPPG) include 5-[4-hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]-3-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-4-oxopentanoic acid and 1-{6-[(1E)-aza-2-(dimethylamino)vinyl]-4-hydroxypyrzolo[5,4-d]pyrimidinyl}-N-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-N-(2-oxypropyl)acetamide. A preferred embodiment of a PPA-containing PNA monomer is 2-[N-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-2-{4-[(4-methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl}acetylamino]acetic acid.
Also provided are methods for the synthesis of oligomers comprising PNA, DNA, RNA and/or chimeras thereof, wherein the aforementioned PNA monomers are used at one or more steps in the synthesis. Oligomers synthesized by these methods are also provided.
In another embodiment, methods for detecting a target sequence in a polynucleotide by hybridization to a probe comprising a DNA, PNA, or PNA/DNA oligomer, wherein one or more residues in the probe comprises a pyrazolopyrimidine or pyrrolopyrimidine base analogue, are provided. In the practice of these methods, the probe can additionally comprise one or more of a ribonucleoside, a fluorophore, a fluorescence quencher and/or a minor groove binder.
In another embodiment, methods for detection of a target sequence in a polynucleotide utilizing compositions comprising a polymeric portion (comprising a polymer) and a fluorogenic portion (comprising one or more fluorophores), wherein one or more purine-containing residues of the polymer are substituted with a residue comprising a pyrazolopyrimidine and/or pyrrolopyrimidine base analogue, are provided. Polymers for use in the method can comprise PNA, DNA, RNA or chimeras thereof, and the base analogue can be present in any of the PNA, DNA or RNA portions of a chimeric polymer. Any number of purine residues in the polymer can be substituted by a base analogue. In a preferred embodiment, the method is practiced using a composition in which a purine residue in the polymeric portion that is directly adjacent to the fluorogenic portion is substituted with a pyrazolopyrimidine or a pyrrolopyrimidine. In another preferred embodiment, oligomers containing three or more consecutive G residues have their consecutive G residues replaced by PPG. Compositions for use in this method can optionally comprise a fluorescence quencher and/or a minor groove binder.
In additional embodiments, methods for detecting a target sequence in an amplification reaction, utilizing the compositions of the invention, are provided. In a preferred embodiment, the amplification reaction comprises a hydrolyzable probe assay.
Also provided are oligomer microarrays wherein at least one of the oligomers described herein is present on the array.
Methods for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a sample, by hybridization to a composition as described herein are also provided. In a preferred embodiment, the target sequence has a single nucleotide mismatch with respect to a related sequence that is also present in the sample, and the composition forms a hybrid with the target sequence but not with the related sequence.